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This protocol describes the procedure or preparing MEF-cultured human pluripotent stem cells (hPSCs) for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. This protocol is to prepare cells for protocol nucleofection of hPSCs. Before starting, familiarize yourself with the protocol and the required preparations. A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" doi: 3. Detailed protocols for preparing plasmids, RNA, and RNP for nucleofection can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above

This collection describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection or electroporation. Collection Overview Preparing MEF-cultured hPSCs for nucleofection Preparing Feeder-free hPSCs for nucleofection Preparing plasmids for nucleofection Preparing mRNA for nucleofection In vitro assembling of RNP for nucleofection Nucleofection of hPSCs Electroporation of hPSCs A. Preparation of MEFs-cultured hPSCs for electroporation B. Preparing plasmids for electroporation C. Electroporation FACS-based enrichment of transfected cells A. Preparation of samples for FACS sorting B. After FACS General notes Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes a standard procedure culturing and transfecting HEK293T cells Protocol overview: A. Culturing HEK293T cells B. Transfection of HEK293T cells with Lipofectamine 2000

This protocol describes the experimental procedure used to measure single cell survival rates post nucleofection of human pluripotent stem cells (hPSCs). General Notes: 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure preparing cell pellets from human pluripotent stem cells (hPSCs) cultured on MEFs for outsourced aCGH karyotyping. General Notes: 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure for staining pluripotency markers, e.g. OCT4, SSEA4, alkaline phosphatase, and etc. on human pluripotent stem cells (hPSCs). Protocol overview A. Immunofluorescence staining B. Alkaline phosphatase staining General notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure preparing feeder-free human pluripotent stem cells (hPSCs) for nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. This protocol is to prepare cells for protocol nucleofection of hPSCs. Before starting, familiarize yourself with the protocol and the required preparations. A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" doi: 3. Detailed protocols for preparing plasmids, RNA, and RNP for nucleofection can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above.

This collection describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection or electroporation. Collection Overview Preparing MEF-cultured hPSCs for nucleofection Preparing Feeder-free hPSCs for nucleofection Preparing plasmids for nucleofection Preparing mRNA for nucleofection In vitro assembling of RNP for nucleofection Nucleofection of hPSCs Electroporation of hPSCs A. Preparation of MEFs-cultured hPSCs for electroporation B. Preparing plasmids for electroporation C. Electroporation FACS-based enrichment of transfected cells A. Preparation of samples for FACS sorting B. After FACS General notes Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes a standard procedure for subcloning of genotype-confirmed human pluripotent stem cells (hPSCs). General notes: 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure of thawing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs). General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. 3. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences.

This protocol describes the procedure FACS-based enrichment of transfected human pluripotent stem cells (hPSCs). Protocol overview A. Preparation of samples for FACS sorting B. After FACS General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Dependent on the genome editing strategy, we use either drug selection or FACS sorting to enrich transfected cell populations based on the presence of a fluorescent protein (either as part of the transfected plasmids or through co-transfection of a fluorescent protein expressing plasmid). 3. Either drug selection or FACS sorting will be usually performed 48 – 72 hours after electroporation/nucleofection. 4. Carefully plan the timeline of FACS experiments and schedule required FACS sorting time at core facility (requires approximately 45 min sorting time per sample) 5. Every FACS sorting experiment requires an additional non-electroporated well of hPSCs (parental cell line) as a negative control to determine the appropriate FACS gates.

This protocol describes the procedure for the in vitro assembly of ribonucleoprotein (RNP) which can be delivered into human pluripotent stem cells (hPSCs) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the thawing of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This collection describes a standard procedure for isolating single human pluripotent stem cell (hPSC) clones in a high-throughput way. This collection follows nucleofection of hPSCs as described in detail in the collection "Nucleofection (Amaxa) andelectroporation(Biorad) of hPSCs;" dx.doi.org/10.17504/protocols.io.b4qnqvve Collection overview Seeding nucleofected hPSCs in 96-well plates using limited dilution Duplicating 96-well plate-cultured hPSCs clones Subcloning of genotype-confirmed hPSCs clones General notes 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure for the preparation of plasmids to be delivered into human pluripotent stem cells (hPSCs) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the process of harvesting and irradiating mouse embryonic fibroblasts (MEFs) to use as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes the freezing of mouse embryonic fibroblasts (MEFs), which can later be used as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. This protocol can be used to freeze MEFs before or after inactivation. 2. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 3. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes a standard procedure for seeding nucleofected human pluripotent stem cells in 96-well plates using limited dilution. This protocol follows nucleofection of hPSCs as described in detail in the collection "Nucleofection (Amaxa) andelectroporation(Biorad) of hPSCs;" dx.doi.org/10.17504/protocols.io.b4qnqvve General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the process of using Mitomycin C to inactivate mouse embryonic fibroblasts (MEFs), which can then be used as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes a procedure for amplifying targeted genomic regions using a PCR reaction

This protocol describes the procedure or preparing MEF-cultured human pluripotent stem cells (hPSCs) for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. This protocol is to prepare cells for protocol nucleofection of hPSCs. Before starting, familiarize yourself with the protocol and the required preparations. A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" doi: 3. Detailed protocols for preparing plasmids, RNA, and RNP for nucleofection can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above

This collection contains protocols which describe the standard procedure of culturing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs). Collection overview Thawing of hPSCs grown on MEFs Passaging of hPSCs grown on MEFs Freezing of hPSCs grown on MEFs A. Freezing of hPSCs as single cell suspension using trypsin B. Freezing of hPSCs as cell aggregates using collagenase General notes 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. 3. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences. 4. While bulk/collagenase passaging is used for routine maintenance of hPSC cultures, manual/microdissection passaging is used to enrich for undifferentiated hPSC colonies (“clean-up” of culture based on undifferentiated hPSC colony morphology) or to expand (“pick”) individual colonies (e.g.,for clonal expansion of individual targeted cells in the process of establishing genome edited cell lines). Manual passaging/ microdissection requires (i) the identification and discrimination of undifferentiated and differentiated hPSC colonies and (ii) the capacity to excise the undifferentiated cells and transfer them to a new plate and can be performed using various approaches as established in many hPSC laboratories.

This protocol describes the standard procedure for preparing mRNA to be delivered into human pluripotent stem cells (hPSCs) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes a standard procedure for duplicating 96-well plate-cultured human pluripotent stem cells (hPSCs). General notes: 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs

This protocol describes the process of using CRISPResso to analyze Next Generation Sequencing (NGS) data from CRISPR-targeted, genome-edited samples.

This protocol describes the standard procedure of using collagenase to passage human pluripotenct stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs). General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. 3. While bulk/collagenase passaging is used for routine maintenance of hPSC cultures, manual/microdissection passaging is used to enrich for undifferentiated hPSC colonies (“clean-up” of culture based on undifferentiated hPSC colony morphology) or to expand (“pick”) individual colonies (e.g.,for clonal expansion of individual targeted cells in the process of establishing genome edited cell lines). Manual passaging/ microdissection requires (i) the identification and discrimination of undifferentiated and differentiated hPSC colonies and (ii) the capacity to excise the undifferentiated cells and transfer them to a new plate and can be performed using various approaches as established in many hPSC laboratories.

This protocol describes a standard procedure for preparing crude cell lysate which can be further analyzed by PCR. Protocol overview: A. Preparing crude cell lysate directly from hPSCs culture B. Preparing crude cell lysate from dissociated cells C. Preparing cell lysate from FACS-sorted cells (small cell number) General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure for the delivery of plasmids into human pluripotent stem cells (hPSCs) using electroporation. Protocol Overview A. Preparation of MEFs-cultured hPSCs for electroporation B. Preparing plasmids for electroporation C. Electroporation General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection. General notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This collection describes a standard genotyping procedure using next generation sequencing (NGS) in the Hockemeyer lab. Collection overview Preparing of genomic DNA from in vitro cultured cells A. Preparing crude cell lysate directly from hPSCs culture B. Preparing crude cell lysate from dissociated cells C. Preparing cell lysate from FACS-sorted cells Amplifying the target genomic region by PCR Preparing samples for NGS CRISPResso analysis General notes: 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the expansion of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes the standard procedure of freezing human pluripotent stem cells (hPSCs), which were grown on inactivated mouse embryonic fibroblasts (MEFs). General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. 3. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences.

This protocol describes the preparation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture. Protocol overview A. Starting with frozen irradiated or Mitomycin C inactivated MEFs (optional) B. Starting with fresh irradiated or Mitomycin C inactivated MEFs General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Either fresh (start at step 4) or frozen stocks of irradiated or Mitomycin C inactivated MEFs can be used to prepare hPSC feeder cells. 3. The indicated MEF density are recommended starting densities and might have to be adjusted for each hPSC line and hPSC medium formulation (KSR, serum-free versus serum-containing medium). 4. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes the procedure of adapting human pluripotent stem cells (hPSCs) to feeder-free culturing conditions using mTeSR-plus or StemFlex General Notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two mediums are not interchangeable. Pick one and stick to it. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex--coated cell culture plates without observing obvious differences.

This protocol describes the process of freezing feeder-free human pluripotent stem cells (hPSCs) using Accutase or ReLeSR Protocol overview: A. Accutase B. ReLeSR General Notes: For this protocol, hPSCs refers collectively to hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex, however these two mediums are not interchangeable. Pick one and stick to it. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex coated cell culture plates without observing obvious differences.

This collection describes the standard procedure of feeder-free culturing of human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex Collection overview Coating plates A. VTN B. Matrigel C. Geltrex Adapting hPSCs cultured on MEFs to feeder-free system Thawing of feeder-free hPSCs Passaging of feeder-free hPSCs A. Accutase B. ReLeSR Freezing of feeder-free hPSCs A. Accutase B. ReLeSR General Notes Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two media are not interchangeable. Pick one and stick to it. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex-coated cell culture plates without observing obvious differences. We have routinely passaged feeder-free hPSCs using either Accutase (as single cell suspension) or ReLeSR (as cell aggregates) without observing obvious differences.

This protocol describes the procedure of thawing feeder-free human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex General Notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two mediums are not interchangeable. Pick one and stick to it. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex--coated cell culture plates without observing obvious differences.

This protocol describes the process of coating plates using either VTN, Matrigel or Geltrex for use in culturing of feeder-free human pluripotent stem cells (hPSCs) Protocol overview Coating plates A. VTN B. Matrigel C. Geltrex General Notes: Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure to knock-in constructs to the AAVS1 safe harbor locus in hPSCs. General Notes: 1. The AAVS1 knock-in construct, AAVS1-SA-neo-CAGGS-PE2-2A-GFP, can be found at AddGene (Catalog: 180014, RRID:Addgene_180014) 2. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.